PB1 (Phox/Bem1p) domains function for specific protein-protein interactions by forming PB1-PB1 domain heterodimers. There are at least 20 human PB1 domain containing proteins. Different PB1 domains contribute to the formation of specific protein complexes for the control of critical biological responses including proliferation, apoptosis, cell polarity, and angiogenesis. These proteins include MEKK2, MEKK3, and MEK5 that are MAP3Ks and the MAP2K, respectively, which solely governs the ERK5 MAPK pathway involved in angiogenesis, cell growth and inhibition of apoptosis. Two additional serine-threonine protein kinases, the atypical PKCs PKC9 and PKC6 also encode PB1 domains. The PKC9 and PKC6 PB1 domains both dimerize with PB1 domains of the scaffold/adaptor proteins p62/Zip (sequestosome-1) and Par6. The p62/Zip adaptor protein is involved in the regulation of NF: B activation and Par6 is a critical scaffold for the control of cell polarization. In addition, p40phox and p67phox use their respective PB1 domains to heterodimerize with each as part of the activation of NADPH oxidase (Nox2), the enzyme complex that catalyzes the generation of reactive oxygen radicals in immune cells. This proposal is based on rapidly accumulating evidence that PB1 domain is a crucial interface for MEKK2-MEK5 interaction involved in various patho-physiological conditions. Inhibition of the PB1-PB1 interaction by small molecules is a unique pharmacological mechanism to selectively disrupt the activation of the EKR5 signaling network in cells. Inhibition of MEKK2-MEK5 PB1-PB1 domain dimerization would be an extremely useful and innovative therapeutic intervention to inhibit angiogenesis, tumorigenesis and potentially chronic inflammation. The aims of this proposal are the development and optimization of robust and cost feasible HTS homogenous TR-FRET screening platforms for identifying small molecule inhibitors of MEKK2-MEK5 PB1-PB1 domain heterodimerization. The initial focus will be to optimize donor- and acceptor- dye conjugated GST MEKK2 or MEK5 fusion proteins and optimization of a robust HTS for 384 or 1536-well format. A counterscreen will be developed with a second PB1-PB1 domain interaction (Par6-aPKCs, p62/Zip- aPKCs, or p40phox-p67phox) that has the potential to multiplexed in the same well with the MEKK2-MEK5 PB1- PB1 domain screen. To validate leads, a live cell FRET-based assay using high content microscopy and straightforward functional cell based pathway assays will be implemented. PUBLIC HEALTH RELEVANCE: Small molecule inhibitors of MEKK2-MEK5 PB1 domain interaction would represent a new target class of drugs for therapeutic intervention to modulate angiogenesis, tumorgenesis, and chronic inflammation. [unreadable] [unreadable] [unreadable]